Lab 4- Column Chromatography and Redox Biochemistry

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May 27, 2024

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Results Part 1: Column Chromatography Table 1. Color of the fractions obtained in Column Chromatography of LDH, Cytochrome C and FMN, and LDH assay results for the clear fractions at 340nm. Fraction Color Rate (Abs/min) at 340 nm LDH activity (μmol/min) % LDH activity #1 Clear 0.04275708 0.007245 5.67% #2 Clear -0.1442514 0.02444 19.1% #3 Clear -0.7537793 0.1277 100% #4 Clear -0.1865322 0.03161 24.8% #5 Light orange -0.02583046 0.004377 3.43% #6 Light orange - - - #7 Orange - - - #8 Brownish orange - - - #9 Light orange - - - #10 Light yellow - - - #11 Light yellow - - - #12 Yellow - - - #13 Bright yellow - - - #14 Yellow - - - #15 Light yellow - - - Part : Reduction using Ascorbate Table 2. Reduction of Cytochrome C and FMN with ascorbate (vitamin C) monitored at the respective wavelengths of 550 nm and 450 nm. Protein Fraction Color Wavelength Absorbance before Ascorbate Absorbance after Ascorbate Flavin Mononucleotide (FMN) #13 Bright Yellow 450 nm 2.0236 1.9819 Cytochrome C #8 Reddish Brown 550 nm 1.0690 1.5765

Calculations  Fraction #1: A = ε x L x C ∆C(mol/min) = ∆A/(ε x L) ∆C(mol/min) = 0.04275708/(6250 x 1) = 6.841 x 10 -6 mol/min NADH (mol/min) = ∆C x Tv = (6.841 x 10 -6 ) x (1.059 x 10 -3 ) = 7.245 x 10 -9 mol/min Enzyme activity =0.007245 μmol/min %LDH activity = (0.007245 μmol/min / 0.1277 μmol/min) x 100% = 5.67 %  Fraction #2: A = ε x L x C ∆C(mol/min) = ∆A/(ε x L) ∆C(mol/min) = 0.1442514/(6250 x 1) = 2.308 x 10 -5 mol/min NADH (mol/min) = ∆C x Tv = (2.308 x 10 -5 ) x (1.059 x 10 -3 ) = 2.444 x 10 -8 mol/min Enzyme activity =0.02444 μmol/min %LDH activity = (0.02444 μmol/min / 0.1277 μmol/min) x 100% = 19.1%  Fraction #3: A = ε x L x C ∆C(mol/min) = ∆A/(ε x L) ∆C(mol/min) = 0.7537793/(6250 x 1) = 1.206 x 10 -4 mol/min NADH (mol/min) = ∆C x Tv = (1.206 x 10 -4 ) x (1.059 x 10 -3 ) = 1.277 x 10 -7 mol/min Enzyme activity =0.1277 μmol/min %LDH activity = 100% because the highest enzyme activity.  Fraction #4: A = ε x L x C ∆C(mol/min) = ∆A/(ε x L) ∆C(mol/min) = 0.1865322/(6250 x 1) = 2.985 x 10 -5 mol/min NADH (mol/min) = ∆C x Tv = (2.985 x 10 -5 ) x (1.059 x 10 -3 ) = 3.161 x 10 -8 mol/min Enzyme activity =0.03161 μmol/min %LDH activity = (0.03161 μmol/min / 0.1277 μmol/min) x 100% = 24.8%  Fraction #5: Column Chromatography and Redox Biochemistry . 2

A = ε x L x C ∆C(mol/min) = ∆A/(ε x L) ∆C(mol/min) = 0.02583046/(6250 x 1) = 4.132 x 10 -6 mol/min NADH (mol/min) = ∆C x Tv = (6.841 x 10 -6 ) x (1.059 x 10 -3 ) = 4.377 x 10 -9 mol/min Enzyme activity =0.004377 μmol/min %LDH activity = (0.004377 μmol/min / 0.1277 μmol/min) x 100% = 3.43% Post-Lab Questions Part 1: 1. In what order did you expect FMN, LDH and Cytochrome C to elute from the column. Explain Why. Gel-Filtration chromatography is conducted to separate the species of proteins or molecules present in the solution according to their size. The column is packed with resin Bio Gel P-200 which contains fine porous beads made of an insoluble polymer. Therefore, the small molecules are capable of entering these porous beads and therefore spend a lot more time in the resin, whereas the larger molecules in the fractionation range of 30-200 kDa, are able to pass through the resin relatively quickly. Thus, this technique allows the large molecules to pass through the column quickly and be the first to elute form the column, whereas the small ones remain in the column chromatography elute the last, since they are in the porous beads longer and travel slower. The Sample mixture provided contained LDH, Cytochrome C and FMN. It was expected that LDH would be the first to elute from the column since it is the largest protein, with a molecular size of 140 kDa and is composed of around 322 residue amino acids. Cytochrome C would be the second and FMN would be the last to emerge out of the column because of their respective sizes of 12kDa and 0.46kDa. 2. Did these three molecules elute in the expected order? If not, suggest a reason why? The three molecules indeed elute in the expected order, with LDH being concentrated in fraction #3, Cytochrome C in fraction #8 and FMN in fraction #13. It was also easy to distinguish the order for cytochrome C and FMN since they are colored reddish brown and yellow respectively. The fractions with the brightest colors were assumed to be the ones with the species being most concentrated in. For LDH, since it is colorless, multiple LDH assays were conducted on the clear fractions and LDH activity was found to be the highest in the fraction #3. 3. What was the purpose of assaying LDH activity in the column fractions? The purpose of assaying LDH activity in the column fractions was to determine which of the fractions had LDH concentrated. And since LDH is colorless, it was difficult to determine its presence in a fraction with naked eye. It was expected that LDH would be the first to be separated since it is the largest protein out of the three, therefore assaying Column Chromatography and Redox Biochemistry . 3

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